Occupational and Environmental Medicine 1995;52:611-620

Biological monitoring of exposure to benzene:a comparison between S-phenylmercapturic acid,trans,trans-muconic acid, and phenol

P J Boogaard, N J van Sittert

Shell Research BV,Shell MolecularToxicology,Badhuisweg 3,PO Box 38000,1030 BN Amsterdam,The NetherlandsP J BoogaardN J van SittertShell InternationalePetroleumMaatschappij BV,Health, Safety, andEnvironmentDivision, BiomedicalServices, PO Box 162,2501 AN The Hague,The Neterlands; andShell BiomedicalLaboratory, ShellNederlandRaffinaderij/ChemieBV, OccupationalHealth and HygieneDivision, PO Box3000, 3190 GAHoogvliet(Rotterdam), TheNetherlandsCorrespondence to:Dr P J Boogaard,Shell Research BV,Shell Molecular Toxicology,Badhuisweg 3, PO Box38000, 1030 BNAmsterdam,The Netherlands

Accepted 15 May 1995

AbstractObjectives-Comparison of the suitabil-

ity of two minor urinary metabolitesof benzene, trans,trans-muconic acid(tt-MA) and S-phenylmercapturic acid(S-PMA), as biomarkers for low levels ofbenzene exposure.

Methods-The sensitivity of analyticalmethods ofmeasuring tt-MA and S-PMAwere improved and applied to 434 urinesamples collected from 188 workers in 12studies in different petrochemical indus-tries and from 52 control workers with nooccupational exposure to benzene. Innine studies airborne benzene concentra-tions were assessed by personal air moni-toring.Results-Strong correlations were foundbetween tt-MA and S-PMA concentra-tions in samples from the end of the shiftand between either of these variables andairborne benzene concentrations. It wascalculated that exposure to 1 ppm (8 hourtime weighted average (TWA)) benzeneleads to an average concentration of 1'7mg tt-MA and 47 jg S-PMA/g creatininein samples from the end of the shift. Itwas estimated that, on average, 3 9%/6(range 1.90/o-7.3%) of an inhaled dose ofbenzene was excreted as tt-MA withan apparent elimination half life of5*0 (SD 2.3) hours and 0*11% (range0-050/%0-26%) as S-PMA with a half lifeof 9 1 (SD 3.7) hours. The mean urinaryS-PMA in 14 moderate smokers and38 non-smokers was 3*61 and 1*99 uglgcreatinine, respectively and the mean

urinary tt-MA was 0 058 and 0 037 mglgcreatinine, respectively. S-PMA provedto be more specific and more sensitive(P = 0-030, Fisher's exact test) than tt-MA. S-PMA, but not tt-MA, was alwaysdetectable in the urine of smokers whowere not occupationally exposed. S-PMAwas also detectable in 20 of the 38 non-

smokers from the control group whereastt-MA was detectable in only nine ofthesesamples. The inferior specificity of tt-MAis due to relatively high background val-ues (up to 0'71 mglg creatinine in thisstudy) that may be found in non-occupa-tionally exposed people.Conclusions-Although both tt-MA andS-PMA are sensitive biomarkers, onlyS-PMA allows reliable determination ofbenzene exposures down to 0 3 ppm (8 hTWA) due to its superior specificity.Because it has a longer elimination half

life S-PMA is also a more reliable bio-marker than tt-MA for benzene expo-sures during 12 hour shifts. For biologicalmonitoring of exposure to benzene con-centrations higher than 1 ppm (8 h TWA)tt-MA is also suitable and may even bepreferred due to its greater ease of mea-surement.

(Occup Environ Med 1995;52:611-620)

Keywords: benzene; biomarkers; S-phenylmercapturicacid; transtrans-muconic acid

The main metabolites that can be found inurine after exposure to benzene are phenoland its conjugates.' As a consequence, totalurinary phenol has been used as a biomarkerfor occupational exposure to benzene for along time. Its use, however, is limited to ben-zene concentrations over 5 ppm (16 mg/M3)as an 8 hour time weighted average (TWA)because a substantial background of phenolmay be found in non-exposed people due todietary habits. In many countries, however,the occupational limit concentration for air-borne benzene will be, or has already been,lowered to 1 ppm (8 h TWA). In some coun-tries even lower values have been adopted(Sweden 0 5 ppm) or proposed (UnitedStates 0 3 ppm). As a consequence new bio-markers are needed. Although measurementof benzene itself either in blood or exhaled airis both highly specific and sensitive,23 urinarymeasurements are preferred in occupationalmedicine as collection of urine is not onlynon-invasive but also easily performed even atdistant working locations. Over the past fewyears two minor urinary metabolites of ben-zene have been introduced as new biomarkersfor determination of benzene exposure:trans,trans-muconic acid (tt-MA, 2,4-hexadi-enedioic acid)4 1 and S-phenylmercapturicacid (S-PMA, N-acetyl-S-phenyl-L-cys-teine)." 14 We applied these two markers in aseries of studies in industrial workers withpotential exposure to benzene to determinetheir comparative suitability for biologicalmonitoring.

Methods and materialsPOPULATIONS AND COLLECTIONS OF URINARYAND AIRBORNE SAMPLESFor the estimation of normal values of urinarytt-MA, a study was conducted in 46 employ-ees from the technical and six employees from

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Summary of studies to monitor benzene exposure

Benzene in air Urinary nA-MA (begin)Number of (mg/Mr3, 8 h TWA) (mg/g creannine)

Working measurementsStudy Activity Country day (workers) Median (Range) Median (Range)

1 Maintenance of North Sea 1 4 (4) 8-4* (<0-1-19-2) 0-06 (0-01-0-23)gas production 2 4 (4) 1-8* (<0-1-3-6) 0-68 (<0-01-3-26)platforms all 8 (4) 1-8* (<0-1-19-2) 0-06 (<0-01-3-26)

2 Maintenance of North Sea 1 3 (3) < 0-1* (<0-13) 0-33 (0-16-0-61)gas production 2 3 (3) 10-1* (7-5-10-7) 0-40 (0 26-0 65)platforms all 6 (4) 3-8* (<0-1-10-7) 0-37 (0-16-0-65)

3 Activities before North Sea 1 11 (11) 0-98* (<0-1-17-9) 0-16 (0-03-1-03)shut down on 2 11 (11) 0-65* (<0-1-15-6) 0-24 (<0-01-1-48)gas production 3 11 (11) 0.33* (<01-3 6) 0 40 (<001-1 88)platform 4 9 (9) 0.10 (0-01-0-24)

all 42 (12) 0-65* (<0-1-17-9) 0-17 (<0-01-1-88)4 Routine operation North Sea 1 4 (4) 0-65* (0 3-2 9) 0-30 (0-06-0 67)

on gas production 2 4 (4) - 0-35 (0-06-1-36)platform 3 4 (4) 2-60* (0.7-6 5) 0-20 (0 06-0 27)

4 4 (4) < 0-1* (< 0-1-1-0) 0-56 (0-30-0-71)all 16 (4) 0-85* (<0-1-6-5) 0-24 (0-06-1-36)

5 Chemical Netherlands 1-5 146 (69) 1-26 (<0-01-211-1) 0-15 (<0-01-0-80)manufacturing

6 Chemical Netherlands 1-5 41 (22) 17t (<0-01-201-5)t -

manaufacturing7 Chemical Germany 1-5 39 (39) -

manufacturing8 Chemical France 1 2 (2) - (0-33-2 28) - (0 05-0 86)

manufacturing 2 2 (2) - (< 0-01-1-95) - (0 08-0-09)3 2 (2) - (0 33-5 20) - (0-01-0-14)4 2 (2) - (0-98-3 25) - (0-01-0-45)5 2 (2) - (< 0-1-0-98) - (0 20-0 20)6 2 (2) - (1-63-1-63) - (0-01-0-07)7 2 (2) - (<01-< 0-1) - (0 52-0 34)8 2 (2) - (<0-1-< 0-1) - (0-01-0-08)all 16 (2) 0-65* (<0-1-5-20) 0-08 (0-01-0-86)

9 Oil refinery with France 5 8 (8)aromatics plant

10 Oil refinery with Germany 1-5 14 (8) 0 77 (0-11-3-30)aromatics plant

11 Road tanker Belgium 1 14 (14) - - 0-08 (0-01-0-40)loading 2 14 (14) - - 0-06 (0-01-0-23)(top filling) 3 14 (14) - - 0-10 (0-01-0-42)

all 42 (14) - - 0-06 (0-01-0-42)12 Pipe line Netherlands 1 2 (2) - (1-3-1-5) - (0-04-0-11)

maintenance 2 2 (2) - (0-8-1-4) - (0-13-0-14)3 2 (2) - - (0-10-0-22)all 6 (2) - (0-8-1-5) - (0-04-0 22)

*12 Hour shifts; personal air monitoring was not performed on all operators.

the medical department who were not occu-pationally exposed to benzene (controls). Thiscontrol group comprised 14 smokers and 38non-smokers.

For the comparison of the methods, 12studies in eight locations (four countries) havebeen performed in the period from 1992 to1994 on workers who were potentiallyexposed to benzene during manufacturingand maintenance operations in natural gasproduction installations, in refineries, and inchemical plants (table).

Four surveys were carried out in 1992 dur-ing various maintenance operations on naturalgas production platforms in the North Sea.

Studies 1 and 2 were both performed ontwo consecutive days, each on four workersinvolved in small maintenance operations.Personal air monitoring was carried out onthese eight workers for the whole 12 hour shiftwith charcoal tubes and personal samplerpumps (flow rate about 100 mi/min). Thedetection limit of airborne benzene was 0-1mg/M3 (12 h TWA). The occupationalhygienist involved noticed skin contact instudy 1 on four occasions (three people). Allthe participants provided urine samples beforeand after the shift for determination of S-PMA and tt-MA (in total 16 samples for study1 and 12 samples for study 2). In the urinarysamples of two operators from study 1 (eightsamples) and three operators from study 2

(seven samples) who had airborne exposure tobenzene higher than 1 5 ppm (12 h TWA)urinary phenol concentrations were alsomeasured.

Study 3 was performed on 16 peopleengaged in activities before shut down. Onlyfive people were involved for one day, theother 11 people were involved for two to fourconsecutive 12 hour shifts. Personal air sam-pling was carried out on these 11 people asdescribed for study 1. All 16 people providedurine samples before and after the shift oneach working day and nine of the 11 peoplewho worked more than one day provided anadditional urine sample 12 hours after the endof the last shift. One worker did not provide asample at the end of the shift on day 3. BothS-PMA and u-MA were determined in all 83urine samples. Also phenol was measured in18 urine samples of workers with airbornebenzene concentrations higher than 2 mg/M3(12 h TWA).

Study 4 was performed on four consecutivedays (12 hour shifts) on four people involvedin routine small maintenance operations.Personal air monitoring was carried out asdescribed for study 1. From all workers urinesamples were collected before and after theshift for measurement of S-PMA and tt-MA(32 samples in total). For two people withrelatively high benzene exposures phenolmeasurements were also made.

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Uninary ti-MA (end) Urinary S-PMA (begin) Urinary S-PMA (end)(mg/g creatinine) (liglg creatinine) (pg/g creatinine)

Median (Range) Median (Range) Median (Range)

4-62 (< 0-01-12-5) < 3 (< 1-5) 151 (< 1-798)1-05 (< 0-01-1-30) 145 (2-496) 35 (3-70)1-05 (<0-01-12-5) 5 (< 1-496) 35 (< 1-798)0-48 (0-13-0-72) 12 (4-104) 15 (3-23)2-59 (2-48-6-23) 20 (14-34) 131 (64-175)1-60 (0-13-6-23) 17 (4-104) 44 (3-175)0-67 (< 0-01-4-06) 3 (< 1-23) 18 (5-392)0-77 (0-14-2-63) 17 (4-143) 15 (1-60)0-37 (0-07-1-28) 19 (5-43) 12 (3-51)- - 7 (2-17) -

0-40 (< 0-01-4-06) 7 (1-143) 12 (1-392)0-35 (0-14-0-76) 3 (2-17) 6 (5-25)0-22 (< 0-01-0-28) 6 (5-49) 4 (< 2-10)1-07 (0-24-1-70) 2 (2-9) 11 (5-66)0-43 (< 0-01-0-85) 6 (5-45) 10 (1-15)0-24 (<0-01-1-70) 4 (2-49) 6 (< 2-66)1-53 (<0-01-20-78) 7 (2-46) 36 (2-2312)

1-61 (0-13-39-49) - 38 (20-1175)

1-08 (0-18-18-18) - 12 (3-277)

- (0-26-0-09) - (2-3) - (11-22)- (0-11-1-97) - (1-12) - (4-66)- (0-01-4-20) - (2-40) - (4-133)- (0-08-2-42) - (1-92) - (3-143)- (0-15-0-81) - (1-31) - (1-41)- (0-74-0-35) - (4-20) - (18-17)- (0-01-0-01) - (21-22) - (9-5)- (0-01-0 05) - (1-5) - (< 2-12)0-13 (0-01-4-20) 5 (1-92) 12 (1-143)0-18 (0-01-0-48) - 26 (3-85)

0-55 (0-11-1-51) - 12 (2-98)

0-17 (0-01-0-53) 1 (< 1-11) 6 (1-13)0-28 (0-08-0-61) 4 (1-14) 5 (1-34)0-20 (0-05-1-05) 5 (1-13) 5 (2-38)0-21 (0-01-1-05) 3 (< 1-14) 6 (1-38)- (0-50-0-57) - (2-4) (3-21)

(0-56-0-57) (2-14) (3-24)- - - (1-21) -

(0-50-0-57) (1-21) (3-24)

Four studies were carried out during rou-

tine operations in chemical plants.Study 5 took place in 1992 in The

Netherlands and involved 69 people whoworked in the production of ethyl benzenefrom benzene. Forty of them were biologicallymonitored only once by measurement of uri-nary metabolites in samples collected both atthe beginning and the end of the eight hourshift (seven people) or only at the end of theshift (33 people). The other 29 people were

biologically monitored on between two to 12different days. In total 169 samples were col-lected and analysed for S-PMA and tt-MA, in115 samples phenol was also measured.Personal air sampling was performed in 44workers for the full shift on one or more days(90 samples in total) with passive dosimeters(3M gas diffusion batches, type 3500). Thedetection limit of airborne benzene was 0 1

mg/M3 (8 h TWA).Study 6 was carried out in the same plant in

1993 on 22 people who provided 41 urine

samples collected at the end of the 8 hourshift on between one and four different days.In all samples tt-MA and S-PMA were deter-

mined, but no measurements of phenol were

made. Personal air monitoring was carried outas in study 5 on eight people (13 samples in

total).Study 7 was carried out in 1993 on 39

workers in a chemical plant in Germany who

handled benzene. Exposure to benzene wasmonitored by measurement of urinary tt-MAand S-PMA. From all workers a single urinesample was collected at the end of the 8 hourshift. No personal air sampling was per-formed.

Study 8 involved two people in a chemicalplant in France who were biologically moni-tored for benzene exposure on eight consecu-tive days by measurement of tt-MA andS-PMA in urine samples from before andafter the shift. In 12 selected samples phenolwas also measured. Personal air sampling wascarried out in all 12 hour shifts, as in study 5.Two studies were carried out in 1992 in oil

refineries with aromatic plants in France andGermany. Study 9 was carried out on eightpeople who collected a single urine sample atthe end of the shift. In the samples tt-MA,S-PMA, and phenol were measured. No airmeasurements were carried out. Study 10 wasperformed on eight workers who providedurine samples at the end of the shift once, onthe last day of the week (two people), ortwice, on the third and fifth day of the five dayworking week (six people). Both tt-MA andS-PMA were measured in all samples, phenolwas measured in three selected samples. Airsampling was by personal sampler pumps(flow rate between 40 and 80 ml/min) withcharcoal as the absorbent. The detection limitwas 0-01 mg/M3 (8 h TWA).

Study 11 was performed in 1992 inBelgium on 14 drivers of road tankers whowere involved in loading the tanker (top fill-ing) with petrols containing an average of 3%v/v of benzene."5 The drivers were biologicallymonitored by measurement of tt-MA andS-PMA in urine samples collected at thebeginning and the end of the 8 hour shift onthree consecutive days. Air monitoring of ben-zene was performed as in study 5. Individualdata were not available.

Study 12 was carried out in TheNetherlands in 1994 on two men involved incleaning and maintenance operations for twodays on a pipeline system used to transportnaphthas with a high content of aromaticcompounds. The men were biologically moni-tored by measurement of tt-MA and S-PMAin urine samples taken before and after theshift and at the beginning of the third day. Airsampling during the activities was carried outwith charcoal tubes and personal samplerpumps (flow rate about 200 ml/min).

In all studies, urine samples were collectedin polythene bottles and acidified to pH 2with 6 M hydrochloric acid on site. Stabilitystudies of tt-MA, S-PMA, and phenol in urinehad shown that under these conditions con-centrations did not change if stored at room

temperature or 40C for at least a month.9 13

The samples were transported to the ShellBiomedical Laboratory, Rotterdam, for mea-surement of urinary benzene metabolites andcreatinine. Urinary creatinine was measuredto adjust for the variations in concentration ofurine samples due to differences in fluidintake between workers. Urinary creatininewas previously shown to provide a good

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correction for spot samples if 24 hour samplescould not be collected'3 and it concomitantlyallows adjustment for the variable dilutionintroduced by the acidification of the samples.Personal air sampling and benzene analyseswere performed by local staff.

MEASUREMENT OF URINARY METABOLITESThe measurement of phenol and creatininehave been described previously.'3 TheS-PMA was also measured as described previ-ously,'3 but as an internal standard deuteriumlabelled S-PMA was used instead ofS-benzylmercapturic acid (S-BMA). The S-[pentadeuterophenyl]mercapturic acid (d5-S-PMA) was synthesised from ring-d5 anilinewith the Gattermann reaction according toRumpPf6 and was recrystallised twice fromethanol with water. The use of d5-S-PMA asan internal standard allowed a limit of detec-tion of 1 jg/l. A series of urine samples werepooled, spiked with S-PMA at two differentconcentrations, acidified, and stored frozen at- 20'C. In all runs a set of samples from thisurine pool was analysed to check the repro-ducibility of the method. The coefficient ofvariation between different runs (n = 42) was8-9% for the pool with 28 pug S-PMA/l and6d1% for the pool with 104 pug S-PMA/l. Thecoefficient of variation of replicate analyses(n = 10) was 2 5% and 3 5% at spiked con-centrations of 13-1 and 50 1 pg S-PMA/l,respectively. With this procedure up to 30urine samples could be analysed in one day.The measurement of tt-MA was essentially

that of Ducos et al.5 In our hands, this methodgave non-reproducible recoveries. It seemedthat the recovery was dependent on both theurinary pH and the brand of the quaternaryammonium ion exchange resin (SAXcolumns) used. With some brands poor recov-eries were obtained with slightly acidic urines.Therefore, the urine samples were brought topH 7-10 by addition of 35% (w/v) aqueoussodium hydroxide solution before the samplewas cleaned up by solid phase extraction. Analiquot of 1I00 ml of urine was subsequentlypassed through a SAX column. The columnwas washed with 3 ml 1% (v/v) aqueous aceticacid and the tt-MA eluted with 4 ml 10%(v/v) aqueous acetic acid into calibrated tubesand the volume was made up to 5 0 ml withpurified water. Aliquots (20 pl) of this solu-tion were analysed by high performance liquidchromatography (HPLC) with UV (A = 259nm) detection (Hewlett Packard 1084B) at20°C with a 100 x 2- 1 mm stainless steel col-umn with Spherisorb 5 ODS-2 as the immo-bile phase and 20% (v/v) methanol in 1%(v/v) aqueous acetic acid as the mobile phase.The flow rate was set at 1 ml/min. For cali-bration, a series of tt-MA standards in freshcontrol urine samples were analysed togetherwith the urine samples. Alkalisation of theurine samples before the solid phase extrac-tion greatly improved the reproducibility:optimum recoveries (invariably over 95%)were obtained if the urinary pH ranged from 7to 10. A more effective clean up was a sideeffect of the alkalisation of the samples, whichlowered the detection limit to 0-01 mg/l. The

coefficient of variation of replicate analyses(n = 10) was 3 2% at a spiked concentrationof 0-64 mg/l and 0-4% at a spiked concentra-tion of 5-55 mg/l. The reproducibility wastested by analysis of a set of identical samplesin a series (n = 6) of runs. The coefficient ofvariation was 11% for samples spiked with1-1 1 mg n-MA/l and 2-7% for samples spikedwith 5-57 mg u-MA/l. With this procedure,up to 40 urine samples could be analysed inone day.

MEASUREMENT OF BENZENE IN AIRThe benzene vapours adsorbed on the gas dif-fusion badges or the charcoal tubes duringpersonal air sampling were desorbed by car-bon disulphide and the solution was analysedwith a gas chromatographic method withflame ionisation detection according to stan-dard methods published by the NationalInstitute for Occupational Safety and Health(NIOSH).'7

CALCULATIONS AND STATISTICSThe cumulative excretions of tt-MA and S-PMA were estimated from the area under thecurve of urinary excretion v time plots andwere used to estimate the percentages of therespiratory benzene dose excreted as tt-MAand S-PMA in the urine. For 14 workers,mainly from studies 3, 4, and 8 personal airmonitoring data as well as sufficient biologicalmonitoring data were available both to esti-mate the respiratory benzene doses and to cal-culate the areas under the curve. For thecalculation of these areas the linear trape-zoidal rule was used in the absorption phasesand the logarithmic trapezoidal rule in theelimination phases. The following formulawas used for the logarithmic trapezoidal rule:

area under the curve(1 2) = 0.0625 x((c - C2) x t2 - t,))/(ln(c,) - ln(C2)),

where 0-0625 is the average urinary volume(1/h), c, denotes the concentration of metabo-lite (either tt-MA or S-PMA) in the urinesample collected at time t, (end of the shift)and c2 the concentration of metabolite at timet2 (next morning) during the eliminationphase (c, > c2). The absorbed benzene dosewas estimated with the formula:

dose = 0-47 x C x V,

where 0 47 is the fraction of benzene that isretained upon inhalation,'8 C denotes the air-borne concentration (mg benzene/m3) and Vthe average respiratory volume (1 25 m3/h).

Both for tt-MA and S-PMA apparent uri-nary elimination rate constants were calcu-lated from workers who had provided urinesamples at the beginning and end of a shift onat least two consecutive days. For the calcula-tion of the apparent elimination rate constants(k-) it was assumed that the absorption wascompleted and that first order eliminationkinetics were obeyed during the collectionperiod. The following formula was used:

ln(c2/c,) = - ke x (t2 - t.),where c, denotes the concentration of meta-bolite (either tt-MA or S-PMA) in the urine

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sample collected at the end of the shift at timet, and c2 the concentration of metabolite atthe beginning of the next day shift at time t2.Occasionally, when the absorption phase wasnot yet completed at the end of the shift (t,),c, was higher than cl. In these cases k, was cal-culated from the decline in metabolite duringthe same shift but only if there was nodetectable exposure to benzene. In these casesc, denotes the concentration of metabolite(either tt-MA or S-PMA) in the urine samplecollected at the beginning of the shift (at timetQ) and c2 the concentration of metabolite atthe end of the same shift (at time t,). For mostworkers values of k, could be calculated morethan once; in these cases the separate valueswere averaged. From the averaged values thecorresponding half life (t,12) was calculated foreach worker with the following formula:

t112= - ln(2)/k,.For all statistical calculations the mainframeversion of the SAS software package (version6-07 or 6&08) was used. For S-PMA and tt-MA results below the calculated value for thelimit of detection of the respective methodshalf of this value was substituted.

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ResultsURINARY tt-MA AND S-PMA IN WORKERS WITHNO OCCUPATIONAL EXPOSUREThe 38 non-smokers in the control group hada mean (SEM) urinary tt-MA concentrationof 0 037 (0-016) and the 14 smokers 0-058(0-013) mg/g creatinine. The mean (SEM) S-PMA concentration in the 38 non-smokerswas 1 99 (0 29) and in the 14 smokers 3X61(0 57) ,ug/g creatinine. S-PMA was present atdetectable concentrations in the urine of allsmokers and in 20 of the 38 non-smokers.This showed the superior sensitivity (P =0-030, Fisher's exact test) of S-PMA overtt-MA, for which urinary concentrations werebelow the detection limit in three smokers and29 non-smokers. Although the delectability(yes, no) of both biomarkers was strongly cor-related with smoking (yes, no) (P < 0000 1,Fisher's exact test), multivariate regressionanalysis disclosed no correlation with thenumber of cigarettes smoked and either S-PMA or tt-MA.

URINARY tt-MA AND S-PMA INOCCUPATIONALLY EXPOSED POPULATIONSThe table gives an overview of the resultsobtained in the 12 studies in industrial workerswith potential exposure to benzene. The highairborne benzene concentrations measured instudies 1, 2, and 3 (up to 19-2 mg/m3) did notreflect normal operations but peak exposuresmeasured in selected operators with relativelyhigh potential exposure to aromatic com-pounds during special maintenance opera-tions on gas production platforms. Bothtt-MA and S-PMA excretion increased overthe shift and reflected the airborne benzeneexposure. During routine operations (study 4)benzene exposure was usually far below1 ppm (3-25 mg/m3) but, nevertheless,increases in both tt-MA and S-PMA werefound. In the urine samples provided by oper-ators with relatively high benzene exposures(> 0-5 ppm) phenol was also measured. Itseemed that phenol correlated well with bothtt-MA and S-PMA in operators with lowbackground values of phenol (< 5 mg/g creati-nine). The very high exposures in studies 5and 6 (up to 211 mg/m3) also representedselected operators who had incidental peakexposures due to accidental spillage duringmaintenance operations. In 68% of thesamples collected in study 5 phenol measure-ments were also carried out. The correlationof phenol with tt-MA as well as with S-PMAwas rather poor: there were several increasedvalues of phenol ( ) 50 mg/g creatinine) with-out concurrent exposure or substantiallyincreased tt-MA or S-PMA, and occasionallyphenol was not increased (s< 5 mg/g creati-nine) although both tt-MA and S-PMA wereclearly increased (tt-MA ) 1 mg/g creatinineor S-PMA > 45 ,ug/g creatinine). Duringoperations in oil refineries with aromaticplants (studies 9 and 10) exposures were gen-erally well below 1 ppm. In all cases distinctincreases in both tt-MA and S-PMA werefound in the samples collected at the end ofthe shift compared with those collected at thebeginning.

Time (h)

Figure 1 Urinay excretion of tn-MA and S-PMA in two workers (A and B) afterexposure to benzene on eight consecutive days.

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URINARY EXCRETION OF tt-MA AND S-PMAOVER TIMEFigure 1 depicts the urinary excretion v timeplots from the two workers from study 8, whowere biologically monitored for eight consecu-tive days. The results of the personal air mea-surements for benzene are shown in thegraphs as bars. The first worker (A) wasexposed to relatively high concentrations ofbenzene up to 5-2 mg/M3 (1-6 ppm) on thethird working day. The second worker wasexposed to much lower concentrations of ben-zene: the highest exposure was recorded onthe sixth working day and was 1-63 mg/m3(05 ppm). It is clear, especially from theexcretion profile of the more highly exposedworker, that the excretion of tt-MA and S-PMA follow roughly the same pattern andthat the excretion of both metabolites reflectthe exposure to benzene. It is also clear that,due to the shorter apparent half life of tt-MAthan S-PMA, tt-MA returns to baseline valuesat the beginning of the next shift after expo-sures up to 1 ppm whereas S-PMA tends toaccumulate at exposures over about 05 ppm.

TOXICOKINETICS OF tt-MA AND S-PMATwenty nine workers provided urine sampleson at least two consecutive days. For 27 work-ers values for the apparent urinary excretionconstants could be calculated from 52 and 38data points for tt-MA and S-PMA, respec-tively. The median k, for tt-MA was 0-136 1/hand for S-PMA 0077 1/h. These values corre-spond to median apparent elimination halflives of 5-1 (mean (SD) 5 0 (2 3)) hours and9 0 (9-1 (3 7)) hours, respectively. For 14workers sufficient personal air monitoring andbiological monitoring data were collected toallow an estimation of the urinary excretion oftt-MA and S-PMA as a percentage of the doseof inhaled benzene. The average percentage of

the dose that was excreted as tt-MA was 3 9%(range 1-9%-7-3%) and as S-PMA was 0-11%(range 0 05%-0 26%).

RELATION BETWEEN DIFFERENT URINARYMETABOLITES OF BENZENEIn fig 2 the tt-MA concentrations were plottedagainst the S-PMA concentration in the sameurine sample, both after logarithmic transfor-mation, for all 188 workers from studies 1-12.If for a worker more than one set of values wasmeasured, the set of values corresponding tothe highest airborne benzene concentrationwas chosen, or if no air measurements wereperformed, the set with the highest values. Ahighly significant correlation between tt-MAand S-PMA was found (r = 0 795, P <0-0001). Nevertheless, some deviations werefound. In some cases where no exposure tobenzene had occurred, relatively high concen-trations of tt-MA (up to 0 71 mg/g creatinine)were measured in the urine (without highconcentrations of S-PMA: < 1 yg/g creati-nine).An exceptional finding was made in study

12. The two workers involved in this studycarried out the same job in close vicinity ofeach other. Figure 3 shows the results of thepersonal air monitoring and the values ofS-PMA and tt-MA. It seems that on the firstday the airborne concentrations of benzeneare more or less identical for both men. Oneof the workers (closed symbols) excreted bothS-PMA and tt-MA in concentrations thatmatch the concentration of benzene in air. Inthe urine of the other man tt-MA was foundas expected, but surprisingly no increases inS-PMA were found. From the medicalrecords it seemed that this worker was treatedfor epilepsy with high doses of carbamazepine,a known inducer of hepatic mixed functionoxidases.

zC

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cn

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Regression line; r= 0.795: P<0.0001given by: log (tt-MA (mg/g creatinine)) =0.836 x log (S-PMA ('g/g creatinine))-1-170

-- 95% Cls (individual)--- 95% Cls (group)

2

-2o 1 2 3

Log S-PMA (,ug/g creatinine)

* tt-MA worker AL tt-MA worker B* S-PMA worker Ao S-PMA worker B

O b, -nAlAA__A .,_1 A- on IvvA benzene worker A ^^-- 8 h TWA benzene worker B E

25 - 1 60 E

C~~~~~~~~~~~C: 20~~~~~~~~~~~~~.C'1 - , 1.20 c

15-

C.)~~~~~~~~~~=. 10 --,

0 0.008 16 24 32 40 48 56 <

Time (h)

Figure 3 Urinary excretion of tt-MA and S-PMA in twoworkers (A and B) after exposure to benzene on twoconsecutive days.

Figure 2 Relationbetween urinaryconcentrations of tt-MAand S-PMA in 188workers exposed tobenzene.

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Biological monitoring of exposure to benzene: a comparison between S-phenylmercapturic acid, trans,trans-muconic acid, and phenol

- Regression line-- 95% Cis (individual)--- 95% Cis (group)

A

200 400 600 800 1000 1200 -0 5 10 15 20 25 30

S-PMA (,ug/g creatinine) tt-MA (mg/g creatinine)

CORRELATION OF AIRBORNE BENZENE ANDURINARY EXCRETION OF METABOLITESThe relation between airborne benzene expo-

sures (8 h TWA) and urinary S-PMA andtt-MA concentrations was examined insamples collected at the end of the shift from58 workers in studies 5, 6, 10, and 12. The

actual working hours in these studies were

close to eight hours. Data from workers whohad worn respiratory protection during themonitoring period were excluded from thestatistics. In cases where more than one datapoint had been generated for a worker, thedata point with the highest airborne benzeneconcentration was used for the linear regres-sion analysis. Highly significant correlations(P < 0-0001) were found for both S-PMAand tt-MA (fig 4). The equations for theregression lines were:

Figure 5 Relationbetween respiratory 12 hTWA exposure to benzeneand concentrations of tt-MA and S-PMA inurinary samples collected atthe end ofa 12 h shiftfrom28 workersfrom studies1-4 and 8.

35 40

benzene in air (mg/m,3 8 h TWA) = 0.0758 x

urinary S-PMA (ug/g creatinine) - 0-317,r = 0-968

benzene in air (mg/m,3 8 h TWA) = 2.38 x

urinary tt-MA (mg/g creatinine) - 0 900,r = 0 959

From the regression lines it was calculatedthat after an 8 h TWA benzene exposure of 1ppm (3-25 mg/M3), the average concentrationin a urine sample collected at the end of theshift would be 47 ug S-PMA/g creatinine and1-74 mg tt-MA/g creatinine. The correspond-ing 95% confidence intervals (95% CIs) forthe group means are 34-61 ,ug S-PMA/g crea-

tinine and 1-2-2-2 mg tt-MA/g creatinine,respectively.

In the other studies where air measure-

ments were made (1-4 and 8) the workers

Regression line-- 95% Cls (individual)--- 95% Cls (group)

20

15

V)E

10E

a)N

c

,I /

/

0 50 100 150 200 250 300 350 400 450 500

S-PMA (,ug/g creatinine)2 4 6 8

Zt-MA (mg/g creatinine)10 12

v

0

Figure 4 Relationbetween respiratory 8 hTWA exposure to benzeneand concentrations of tt-MA and S-PMA inurinary samples collected atthe end ofan 8 h shiftfrom58 workersfrom studies 5,6, 10, and 12.

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were on duty in 12 hour shifts. A separateregression analysis was made with the datacollected in these studies. Again, only one setof data was included for each worker and datacollected while respiratory protection wasworn were excluded. Also, data collected dur-ing operations with apparent dermal expo-sure, as recorded by the occupationalhygienist, were excluded from the analyses.Finally, the following regression equations(fig 5), with highly significant correlations(P < 0-0001), were obtained by analysingdata sets from 28 workers:

benzene in air (mg/m,3 12 h TWA) = 0-0507x urinary S-PMA (ug/g creatinine) + 0-518,

r = 0 959

benzene in air (mg/M,3 12 h TWA) = 1.98 xurinary tt-MA (mg/g creatinine) + 0 390,

r = 0-862

DiscussionBecause of the potential adverse healtheffects, including acute myeloid leukaemia,that have been associated with long termoccupational exposure to high concentrationsof benzene (100-500 ppm), it is important tominimise exposure to benzene. To evaluatethe effectiveness of control measures, biologicalmonitoring techniques are needed that allowdetection of benzene to well below 1 ppm.Due to the high and variable background ofurinary phenol caused by endogenous pro-duction of phenol as well as high dietary andenvironmental exposure to phenol, urinaryphenol is not a suitable variable for biologicalmonitoring of exposure to benzene of below5 ppm. In the past few years, tt-MA and S-PMA, two minor metabolites of benzene,have been proposed to replace phenol as bio-markers of exposure to benzene. Thesemetabolites can both be measured in urine,which in our view is a prerequisite for goodbiological monitoring. Since its introductionin 1985, improved methods have been pub-lished for the measurement of urinary tt-MAby Inoue and co-workers4 who claimed to beable to detect benzene exposure to 6-7 ppmand by Ducos and co-workers5 6 whoimproved Inoue's method by increasing thesensitivity, which allowed biological monitor-ing of benzene exposure down to 1 ppm (8 hTWA). Recently, we published an improve-ment of the method for the determination ofS-PMA as proposed by Stommel and co-workers'2 that allows routine biological moni-toring of exposure to benzene as low as 0 3ppm (8 h TWA).'3

This paper reports the results of 12 sepa-rate investigations where measurements ofboth urinary tt-MA and S-PMA were applied.The methods were compared for sensitivity,specificity, and ease of operation, to get a bet-ter insight into their suitability for routine bio-logical monitoring of industrial exposure tolow concentrations of benzene. To a limitedextent urinary phenol measurements werealso performed to enable a comparison of this

established biological monitoring methodwith the more recent biological monitoringmethods.We found that measurement of tt-MA as

described by Ducos and coworkers sometimesgave non-reproducible results.56 It turned outthat this phenomenon was due to differencesin the quaternary ammonium ion exchangecolumns. With some brands the recovery of tt-MA from the columns seemed to be depen-dent on the pH of the urine sample.Alkalisation of the samples to a pH of 7 to 10gave optimum recoveries that were invariablyover 95%. It seemed that the solid phaseextraction was more efficient at a higher pH,which resulted in cleaner extracts and a lowerlimit of detection (0-01 mg/l). Recently, thissame favourable effect of alkalisation was alsoreported by Lee and coworkers who added 2volumes of a TRIS buffer (pH 10) to theurine samples before the solid phase extrac-tion.9 Overall, the method proved to besimple, straightforward, and robust, whichresulted in small variation coefficients withinand between runs.

In the method used for the determinationof S-PMA, the internal standard S-BMA wasreplaced by deuterated S-PMA in the presentstudy for two reasons. The first reason was toimprove the sensitivity and reliability of themethod. In our previous report it was statedthat the limit of detection varied from 1 to5 jug/l, depending mainly on matrix effects.'3 Itseemed that in urine specimens with a highspecific gravity, S-PMA concentrations lowerthan about 5 ,ug/l could in certain cases not beattributed with certainty. With deuterated S-PMA as the internal standard this problemwas solved and a limit of detection of 1 ,ug/lcould be guaranteed.The second reason was that toluene was

recently reported to be metabolised inhumans to S-BMA. 19 20 In many industrialprocesses with a risk of exposure to benzeneco-exposure to toluene may occur and bio-transformation of toluene to S-BMA mightinterfere with its use as an internal standard.In some preliminary studies we could indeedidentify small amounts of S-BMA (up to 4 0,ug/l) in 10 workers exposed to low concentra-tions of toluene (up to 5-3 ppm, 8 h TWA)(unpublished observations). We found in thisas well as in our previous studies'3 that only0-11% of inhaled benzene is metabolised toS-PMA and although toluene is probablymetabolised even less to S-BMA, there mightstill be serious interference in some cases asthe airborne concentrations of toluene mayexceed those of benzene by one or two ordersof magnitude in many industrial operations.The problem of interference due to co-expo-sure to toluene may also be overcome with ahalogenated phenylmercapturic acid, such asS-4-fluorophenylmercapturic acid, as theinternal standard,'2 1" but this will not solvethe loss of sensitivity due to matrix effects.

Because of the increased sensitivity of themeasurement of S-PMA, the benzene uptakedue to cigarette smoking was detectable in all14 smokers from the control group (mean

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(SEM) 3-61 (0-57) ,ug/g creatinine). In 20 outof the 38 non-smokers of the control groupS-PMA could also be detected albeit inmuch lower concentrations (1 99 (0 32) ,ug/gcreatinine). The sensitivity of the tt-MAmeasurement was too low to allow detectionof tt-MA in most non-smokers and somesmokers in the control group. The concentra-tions of tt-MA in the smokers (0-058 (0-013)mg/g creatinine) and non-smokers (0 037(0-16) mg/g creatinine) were relatively lowcompared with the results of a recent study ontt-MA conducted in Singapore where valuesof 0-19 and 0-14 mg/g creatinine were foundin 35 smokers and 23 non-smokers, respec-tively.9 Although the same detection limit fortt-MA was reported as in our study, tt-MAwas detectable in all smokers and almost halfof the non-smokers. The investigators thoughtthat their relatively high background valueswere due to environmental pollution. Inanother recent study values of 0-29 and 0 09mg tt-MA/g creatinine were measured in 42smokers and 42 non-smokers, respectively.Although the smokers in these two studiesseemed to consume larger quantities of ciga-rettes than the moderate smokers in our con-trol group, there was no correlation betweenthe number of cigarettes smoked and excre-tion of tt-MA,9 but a weak correlation wasfound between excretion of tt-MA and urinarycotinine concentrations.The toxicokinetics, based on spot samples,

were studied for both tt-MA and S-PMA. Themean (SD) apparent urinary elimination halflife (ti,,) for S-PMA was 9 1 (3.7) hours,which is almost identical to the t,12 of 9 0 (4 5)hours previously reported.'3 No discrete val-ues for the tl,2 of tt-MA have been reported asyet, but it has been found that in humans tt-MA showed a t1,2 similar to that of phenol.5Our data corroborate this finding: the esti-mated tl/2 for tt-MA was 5 0 (2-3) hours. Theaverage percentages of the doses of inhaledbenzene that were excreted in the urine as tt-MA and S-PMA were 3 9% (range1-9%-7-3%) and 0-11% (range 0 05%-0 26%), respectively. Our value for the excre-tion of tt-MA is about twice the only value of1-9% reported so far.4 This difference is partlyexplained by the lower respiration rate of 09m3/h used for the estimation by Inoue andcoworkers. The value found for S-PMA con-firms our previous findings of a mean excre-tion of 0 1 1% as S-PMA with a range of 0 05to 0 29%.'3 Most probably these rather wideranges for both tt-MA and S-PMA are partlydue to the fact that the calculations are basedon spot samples, with the inherent risk ofunder and overestimation of areas under thecurve.

Despite the fact that only spot sampleswere collected and that tl/2 of tt-MA is consid-erably shorter than that of S-PMA a goodcorrelation was found between urinary con-centrations of tt-MA and S-PMA (fig 2). Alsogood correlations between benzene in air andS-PMA as well as tt-MA were found (figs 4and 5). The 95% CIs in these figures as wellas the correlation coefficients for the regres-

sion lines indicate that the reliability of tt-MAand S-PMA as biomarkers for benzene expo-sure during eight hour shifts are almost identi-cal. It is obvious, however, that the reliability oftt-MA as a biomarker for benzene exposureduring 12 hour shifts is much less than forS-PMA, which is totally due to its shorterelimination half life.From these correlations it follows that

exposure to 1 ppm benzene (8 h TWA) willlead to an average excretion of 1 74 mg tt-MA/g creatinine and 47 ug S-PMA/g creati-nine in urine samples collected at the end ofthe shift. These values seem to be confirmedby the regression equation in fig 2, fromwhich it can be calculated that 47 pg S-PMA/g creatinine corresponds to 1 69 mg tt-MA/g creatinine. The value of 47 Pg/gcreatinine for S-PMA also agrees well with thefindings from our previous studies, where asimilar value was found.'3 Bechtold and co-workers found in a group of 14 Chinesefemale workers that exposure to 4-4 ppm ben-zene (8 h TWA) led to an average excretion of6-2 mg tt-MA/g creatinine in urine samplescollected at the end of the shift. This matchesour results extremely well as according to theregression line obtained with our data such anexposure would lead to excretion of 6-4 mg/gcreatinine. Our findings are also in goodagreement with two other studies in 23French and 38 Belgian male workers where itwas found that exposure to 1 ppm benzene(8 h TWA) corresponded to 1 mg tt-MA/16and 1-4 mg n-MA/g creatinine,'0 respectively.

In some cases skin contact with liquids con-taining benzene was reported by the industrialhygienist. The personal air monitoring dataand the concentrations of tt-MA and S-PMAmeasured in the urine collected at the end ofthe shift strongly suggest that in these casesrather extensive dermal uptake of benzenehad taken place. For example, during thereplacement with bare hands of a plunger in askimmerpump for natural gas condensate instudy 1, an airborne benzene concentration of19-2 mg/M3 was measured as a 12 h TWA. Inthe urine collected at the end of the shift 12-5mg tt-MA and 798,ug S-PMA/g creatininewere measured. According to the regressionequations for the relation between airbornebenzene (12 h TWA) and the urinary meta-bolites in urine at the end of the shift, the con-centration tt-MA and S-PMA would indicateexposure to 25-2 and 41-0 mg benzene/ml,respectively. Although the value for tt-MA isjust inside the 95% CI for individual results, itis extremely unlikely (P < 0-001) that theresult for S-PMA is solely due to inhalationexposure.The present study was undertaken to com-

pare the suitabilities of tt-MA and S-PMA forbiological monitoring of low level benzeneexposure. Our results confirm previous find-ings by ourselves and others that the measure-ments of both urinary tt-MA and S-PMA aresensitive tests for monitoring exposure to lowconcentrations of benzene. We confirmed thesuitability of S-PMA to detect exposures tobenzene as low as 0 3 ppm (8 h TWA). This

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low level is fully due to the high specificity ofthe S-PMA test. The increased sensitivityachieved by lowering the detection limit led topossible detection of exposure to benzenecaused by moderate smoking. Despite the factthat we were able to improve the method ofmeasurement of urinary tt-MA by a more effi-cient clean up, we also confirmed the conclu-sions by others that with tt-MA, at best,exposures of over 05 ppm can reliably bedetected.'014 The reason for this is that inunexposed people relatively high backgroundconcentrations of tt-MA are consistentlyfound.8 0112' This background may possiblybe due to consumption of sorbic acid, whichis used as a preservative in many foodstuffs.5There may be a preference for uA-MA overS-PMA as a biomarker for benzene exposuresof more than 1 ppm (8 h TWA) as HPLC iseasier to operate than gas chromatography-mass spectroscopy and in most cases is alsomore readily available. Due to the ratherexpensive solid phase extraction, the cost/sample of analysis of tt-MA is about equal tothat of S-PMA. A disadvantage of tt-MA forbiological monitoring of exposure to benzeneis that its formation is decreased in the eventof co-exposure to other aromatic hydro-carbons,22 which may lead to underestimationof benzene exposure. Although we have notyet been able to confirm it, our findings instudy 12, where hardly any S-PMA was foundin a man treated with high doses of an inducerof the hepatic mixed function oxidase, mightindicate that the formation of S-PMA may beinfluenced by co-exposure as well.

The technical assistance by H van der Waal, J Kweekel, andY N Vreugd (Shell Biomedical Laboratory; Shell NederlandRaffinaderij/Chemie B V, Rotterdam, The Netherlands) isgratefully acknowledged.

1 Yardley-Jones A, Anderson D, Parke DV. The toxicity ofbenzene and its metabolism and molecular pathology inhuman risk assessment. BrJIndMed 1991;48:437-44.

2 Angerer J, Scherer G, Schaller KH, Muller J. The determi-nation of benzene in human blood as an indicator ofenvironmental exposure to volatile aromatic com-pounds. Fresenius Journal of Analytical Chemistry 1991;339:740-2.

3 Brugnone F, Perbellini L, Maranelli G, Romeo L,Guglielmi G, Lombardini F. Reference values for bloodbenzene in the occupationally unexposed general popu-

lation. Int Arch Occup Environ Health 1992;64:179-84.4 Inoue 0, Seiji K, Nakatsuka H, Watanabe T, Yin SN, Li

GL, et al. Urinary tt-muconic acid as an indicator ofexposure to benzene. BrJ'Ind Med 1989;46:122-7.

5 Ducos P, Gaudin R, Robert A, Francin JM, Maire C.Improvement in HPLC analysis of urinary transtrans-muconic acid, a promising substitute for phenol in theassessment of benzene exposure. Int Arch Occup EnvironHealth 1990;62:529-34.

6 Ducos P, Gaudin R, Bel J, Maire C, Francin JM, RobertA, et al. transtrans-Muconic acid, a reliable biologicalindicator for the detection of individual benzene expo-sure down to the ppm level. Int Arch Occup EnvironHealth 1992;64:309-13.

7 Bechtold WE, Lucier G, Birnbaum LS, Yin SN, Li GL,Henderson RF. Muconic acid determinations in urine asa biological exposure index for workers occupationallyexposed to benzene. Am Ind HygAssocJ 1991;52:473-8.

8 Johnson ES, Lucier G. Perspectives on risk assessmentimpact of recent reports on benzene. Am _J Ind Med1992;21:749-57.

9 Lee BL, New AL, Kok PW, Ong HY, Shi CY, Ong CN.Urinary transtrans-muconic acid determined by liquidchromatography: application in biological monitoring ofbenzene exposure. Clin Chem 1993;39:1788-92.

10 Lauwerys RR, Buchet JP, Andrien F. Muconic acid inurine: a reliable indicator of occupational exposure tobenzene. Am I IndMed 1994;25:297-300.

11 Rauscher D, Lehnert G, Angerer J. Biomonitoring ofoccupational and environmental exposures to benzeneby measuring transtrans-muconic acid in urine. ClinChem 1994;40:1468-70.

12 Stommel P, Muller G, Stucker W., Verkoyen C, Schobel S,Norpoth K. Determination of S-phenylmercapturic acidin the urine-an improvement on the biological moni-toring of benzene exposure. Carcinogenesis 1989;10:279-82.

13 Van Sittert NJ, Boogaard PJ, Beulink GDJ. Application ofthe urinary S-phenyimercapturic acid test as a biomarkerfor low levels of exposure to benzene in industry. Br 7IndMed 1993;50:460-9.

14 Popp W, Rauscher D, Muller G, Angerer J, Norpoth K.Concentrations of benzene in blood and S-phenylmer-capturic acid and t,t-muconic acid in urine in carmechanics. Int Arch Occup Environ Health 1994;66:1-6.

15 CONCAWE's petroleum products and health manage-ment groups. Gasolines. Product dossier 921103. Brussels:CONCAWE, 1992.

16 Rumpf P. Darstellung, Charakteriserung und Trennungder Merkaptursauren von Benzol, Toluol, Ethylbenzolund Xylolen durch GC-MS Analyse. Hamburg:Universitgt Hamburg, 1981. (PhD Thesis.)

17 NIOSH. Back up data report benzene, method S311. Manualof analytical methods, 2nd ed. Vol 3. Cincinnati: DHEW(NIOSH), 1977. (Publication No 77-157C.)

18 Doctor JH, Zielhuis R. Phenol excretion as a measure ofoccupational exposure. Ann Occup Hyg 1967;1O:317-36.

19 Takahashi S, Matsubara K, Hasegawa M, Akane A,Shiono H. Detection and measurement of S-benzyl-N-acetylcysteine in urine of toluene sniffers using capillarygas chromatography. Arch Toxicol 1993;67:647-50.

20 Takahashi S, Kagawa M, Shiwaku K, Matsubara K.Determination of S-benzyl-N-acetyl-L-cysteine by gaschromatography/mass spectrometry as a new marker oftoluene exposure. JAnal Toxicol 1994;18:78-80.

21 Melikian AA, Prahalad AK, Hoffmann D. Urinarytrans,trans-muconic acid as an indicator of exposureto benzene in cigarette smokers. Cancer EpidemiolBiomarkers Prev 1993;2:47-51.

22 Inoue 0, Seiji K, Watanabe T, Kasahara M, Nakatsuka H,Yin S, et al. Mutual metabolic suppression between ben-zene and toluene in man. Int Arch Occup Environ Health1988;60:15-20.

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